RNA-Seq of Tinospora sagittata in response to excessive and deficient nitrogen fertilizer
There are two main ways to obtain mRNA: one is to utilize the structural characteristics of most mRNA in eukaryotes with polyA tails, and enrich mRNA with polyA tails through Oligo (dT) magnetic beads; The second is to remove ribosomal RNA from the total RNA to obtain mRNA. Subsequently, a fragmentation buffer was added to break the RNA into short segments. Using the short segment RNA as a template, the first stranded cDNA was synthesized using six base random primers. Then, buffer, dNTPs (dTTP, dATP, dGTP, and dCTP), and DNA polymerase I were added to synthesize the second stranded cDNA. Subsequently, the double stranded cDNA was purified using DNA purification magnetic beads. The purified double stranded cDNA is then subjected to end repair, followed by adding an A-tail and connecting sequencing adapters. Then, DNA purification magnetic beads are used for fragment size selection, and PCR enrichment is performed to obtain the final cDNA library.